JP2002128784A - Cell-invasive excited singlet oxygen generating compound and drugs containing the compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent which invades a eukaryotic cell, generates singlet oxygen thermally at the cell, particularly at body temperature, and kills the cell. A cell-invasive agent that generates singlet oxygen at body temperature, comprising a compound represented by the formula: Embedded image In the general formulas A, B and C, at least one of R ₁ , R ₂ , R ₅ and R ₆ , at least one of R ₉ , R ₁₀ , R ₁₃ and R ₁₄ and at least one of R ₁₅ to R ₁₈ One is CO
OR ₂₁ (where R ₂₁ is a lower alkyl group).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a method for invading a cell,
The above general formulas A and B, which are used as agents that generate singlet oxygen thermally in the cells, particularly at body temperature.
And a singlet oxygen generating compound represented by C.

[0002]

2. Description of the Related Art Singlet oxygen is an active chemical species, and it is known that singlet oxygen is used as a chemical species having a bactericidal action such as treatment of cancer or killing a virus attached to foods. It is. In this situation, much attention to toxicity have been given for the reaction and viable cells singlet oxygen ^{_{(1 O 2, 1 Δ g}} ) of an organic compound. Known NaOCl-
H ₂ O _2, myeloperoxidase -H ₂ O ₂ - halogen ionic, and light - singlet oxygen generating system such as photosensitizers -O ₂ system, the reactive oxygen species to another singlet oxygen Contain, and often produce free radicals, so that such systems were not available as pure singlet oxygen evolution reactions in either extracellular or intracellular systems. Above all, light-
Photosensitizer-O ₂ system is used for photodynamic therapy (pho
called todynamic therapy)
For example, by giving a hematoporphyrin derivative and using a fiber optic to irradiate a laser beam to a lesion formed of a tumor in which the dye is incorporated, singlet oxygen can be generated to kill tumor cells. It is in the spotlight for being able to do it.

On the other hand, the compound 3,3 '-(1,1,2) which is triggered by the generation of singlet oxygen completely different from that described above, that is, is not photochemical, but is activated by heat as a trigger. 4-naphthylidene) dipropionic acid disodium salt and the like have been reported [Paolo Di Mascio, Helmut Si
es, J. Am. Chem. Soc., Vol. 111, No. 8, pp. 2909-2914 (198
9)] Furthermore, Saito et al. Reported the synthesis of a novel water-soluble naphthalene endocyclic (NEPO) that generates active oxygen at the physiological pH triggered by heat [Isao Saito, Teruo Matsuura, Kenzo
Inoue, J. Am. Chem. Soc., Vol. 103, No. 1, pp. 188-190
(1981)]. Recently, Nakano, Fujimori et al. Reported that wild-type E. coli was exposed to the NEPO.
Adenosine triphosphate (A) by respiratory enzyme system in cell membrane
TP), the Escherichia coli was easily killed, whereas eukaryotic cells having a respiratory enzyme system in the cell membrane but not in the cell membrane were not damaged by singlet oxygen [H . Tatsuzawa, T. Maruyama, N. Misawa, K. Fujimor
i, K. Hori, Y. Sano, Y. Kambayashi, M. Nakano, FEB
S Letters, Vol.439, pp.329-333 (1998)]. This means that the singlet oxygen lifetime in the cell system is estimated to be on the order of nanoseconds, so even if NEPO in the extracellular fluid thermally generates singlet oxygen efficiently, the singlet oxygen remains in the cell. It suggests that it cannot reach and attack the intracellular mitochondrial respiratory chain enzyme system because it is deactivated and changes to normal ground state oxygen molecules before entering. That is, in a eukaryotic cell system that does not have a respiratory enzyme system in the cell membrane and has intracellular mitochondria, as long as singlet oxygen cannot reach the respiratory system in the cell, the cell killing action by such a mechanism is Understood as not working. The above indicates that the bacterial and possibly mitochondrial respiratory chain systems involved in the formation of adenosine triphosphate (ATP) are very susceptible to singlet oxygen attack.

[0004]

An object of the present invention is to utilize singlet oxygen to kill cells such as tumors. The singlet oxygen generation trigger mechanism of Saito et al. An object of the present invention is to construct a system that can be expressed in a eukaryotic cell line that has no respiratory enzyme system but has intracellular mitochondria. In order to construct such a system, it is necessary to design a compound to be taken into cells while maintaining the singlet oxygen generation structure while having the trigger mechanism.
The present inventors, while studying a compound having the above properties, basically has sufficient hydrophobicity to have cell membrane permeability to a compound having a singlet oxygen generation trigger mechanism such as Saito, and, After infiltration into the cell, it is thought that it is good to create a drug that is rapidly hydrolyzed by the action of intracellular esterase and changes into a water-soluble singlet oxygen generating agent accessible to mitochondria in the cell, and the general formula A, The compounds of B and C were synthesized to solve the above-mentioned problems of the present invention. By injecting this into the breast cancer cells via the esophagus and to reach the liver cancer lesion, it becomes possible to effectively kill the cancer cells in the lesion. Moreover, the compounds of the general formulas A, B and C have a short half-life of 30 minutes and a short duration of cytotoxicity under the conditions of the singlet oxygen generation, and therefore have low toxicity to normal cells. The singlet oxygen generated therein kills prokaryotic cells but has very low toxicity to eukaryotic cells, and has the advantage of being easily used as a therapeutic agent.

[0005]

According to the present invention, there is provided a cell-invasive singlet oxygen generating compound represented by the above general formula A, B or C, and a singlet oxygen generating compound at a body temperature comprising the compound. It is a cell-penetrating drug that occurs. Preferably, an injection containing the compound represented by the general formula A, B or C, or the general formula A, B
Alternatively, the medicament is characterized in that the medicament containing the compound represented by C is held in a sustained-release carrier.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail.
A. In the general formula A, m and n are 1, R ₁ = CO
OCH ₃ , a compound in which R ₂ to R ₈ are H (compound M) is synthesized by the method described below. Other general formulas A,
The compound represented by B or C can be synthesized in the same manner as the compound M. 1,3- (4′-methyl-
1'-Naphthyl) -propionic acid, 1 ', 4'-endoperoxide (NEPO) was synthesized according to the method described in the literature (K. Fujimori ,, T. Komiyama, H. Taba).
ta, T. Nojima, K. Ishiguro, Y. Sawaki, H. Tatszaw
a, M. Nakano, Photochem. Photobiol. Vol.68 (No.2), p
p.143-149 (1998)). The compound obtained by alkyl esterification, particularly methyl esterification of the NEPO compound is synthesized by the following method (synthesis method A).

Synthetic method A 3- (4'-methyl-1'-naphthyl) propionic acid methyl ester [Methyl 3- (4'-methyl-1'-naphthyl) -propionate]
Synthesis of 3- (4'-methylnaphthyl) propionic acid [3- (4'-methyl-1 '
-naphthyl) -propionicacid] was added dropwise to a solution prepared by dissolving 1.0 g in 5 ml of ether while stirring, until nitrogen bubbles did not appear and the yellow color of diazomethane did not disappear. The diazomethane ether solution was prepared by the method of F. Arnet (Organic Synthesis, Collective V
olume 2, 165-167, (1966)).
After confirming that the starting carboxylic acid had disappeared by thin layer chromatography, the solution was subjected to a silica gel column (φ20 m
m, length 40 mm). The column was washed with ether until the target compound was no longer eluted with ether, a fraction containing the target compound was collected, and the ether was distilled off. The obtained colorless oil was dissolved in a small amount of methanol, and allowed to stand in a refrigerator at −20 ° C. overnight, whereupon colorless crystals precipitated. The methanol solution was removed with a drop, and the remaining crystals (melting point: below room temperature) were washed with methanol at -20 ° C. Then, the remaining methanol was removed by a vacuum pump, and 0.72 g of a colorless oily methyl 3- (4'-met) was removed.
hyl-1'-naphthyl) -propionate was obtained. It was confirmed to be a single component by thin layer chromatography.

Synthesis of NEPO methyl ester (Compound M) a. Synthesis method 1 Methyl 3- (4'-methyl-1'-naphthyl) -propionate 0.72
And 3 mg of methylene blue in 20 ml of dichloromethane were added to a reaction flask having three long necks. A glass tube filled with silica gel particles was attached to one opening, and a glass tube for oxygen gas bubble was inserted into the other opening. The reaction flask was immersed in a Dewar containing liquid nitrogen-ethanol cryogen at −10 ° C. and having a light transmission window, and 500 W was externally placed in a Pyrex tube while oxygen gas was bubbled through the solution.
Light irradiation was performed with a halogen lamp. During the photosensitized oxygenation reaction, the Dewar bottle containing the reaction vessel and the glass tube containing the halogen lamp were immersed in running water in a plastic bucket and cooled. The progress of the reaction was checked at any time by thin-layer chromatography, and methylene blue was replenished. After 25 hours, when the disappearance of the raw materials was recognized, the photosensitized oxygenation reaction was stopped. Chromatographic column with jacket (20 mm ID)
Is filled with 25 mm long silica gel and -20
The reaction solution was poured onto the column while circulating liquid nitrogen-ethanol cryogen at ℃. Further, dichloromethane was passed through the column, and the distillate was collected in a container kept at -20 ° C. The fractions containing the desired NEPO methyl ester were confirmed by thin layer chromatography, and the solvent was distilled off under reduced pressure at a temperature of 0 ° C. or lower to obtain 0.82 g of a colorless oily NEPO methyl ester. b. Synthesis method 2 To a solution of 0.50 g of NEPO synthesized by a known method dissolved in 30 ml of ether at 0 to -5 ° C, add an excess amount of a diazomethane ether solution, and leave at 0 to -5 ° C for 1 hour. Low-temperature silica gel column chromatography was performed in the same manner as described in Method 1 to obtain 0.52 g of NEPO methyl ester.

B. Kinetics of Thermal Decomposition (Singlet Oxygen Generation) of NEPO Methyl Ester As the following measurement solvent, ethanol or 0.1 M phosphate buffered saline containing 1% ethanol (PBS = phosphate-b)
uffered saline, pH 7.4) was used. 3- generated when singlet oxygen is released from NEPO methyl ester
When the absorption spectrum of (4'-methyl-1'-naphthyl) -propionic acid methyl ester (NPA methyl ester) was measured with a spectrophotometer (Type: U-3210, manufactured by Hitachi, Ltd.), the absorption peak was 288 nm. Was. The singlet oxygen release and decomposition rate of NEPO methyl ester at 37 ° C. is 10-20 μM NEPO methyl ester solution 3 m
L was placed in a UV absorption measurement cell made of quartz and determined by measuring the time-dependent change of 288 nm intensity based on NPA methyl ester.

The time course of NEPO methyl ester degradation (NPA methyl ester formation process) in PBS at 37 ° C. in 1% ethanol follows a linear function (FIG. 1). FIG. 1 shows the time course of the formation of NPA methyl ester from NEPO methyl ester at pH 7.4. That is, a solution obtained by adding about 10 μM NEPO methyl ester to 3 mL of PBS containing 1% ethanol at pH 7.4 was incubated at 37 ° C., and the solution derived from NPA methyl ester derived from NPA methyl ester was incubated at 37 ° C. for the time shown in FIG.
The change in absorbance at 88 nm was followed. A ₀ and A _max are N
The initial value and the maximum absorption value of the absorbance at 288 nm derived from PA methyl ester are shown. The inset in FIG.
It is the spectrum of the NPA methyl ester measured in PBS containing 1% ethanol in H7.4 as a solvent. NE
PO methyl ester is thermally decomposed according to the following reaction formula 1 to produce NPA methyl ester and singlet oxygen ( ¹ O ₂ ), or NPA and singlet oxygen ( ¹ O ₂ ) by hydrolysis and thermal decomposition. .

[0011]

Embedded image

The first-order rate constant (k) of the reaction is calculated from FIG. 1 as (4.32 ± 0.28) × 10 ⁻⁴ s ⁻¹ . This value is N
It is the same as the first-order rate constant of the thermal decomposition reaction of EPO (4.16 × 10 ⁻⁴ s ⁻¹ ). K in ethanol is also (1.73 ± 0.3
1) × 10 ⁻⁴ s ⁻¹ and the decomposition rate constant of NEPO (1.64 × 10 ^{−4 in} methanol; 1.55 × 10 ^{−4 in} chloroform)
s ^-1 ). The molar extinction coefficient (ε) of NPA methyl ester in ethanol and in PBS containing 1% ethanol was 8,600 M ⁻¹ c, respectively.
m- ¹ and 6,660 M- ¹ cm- ¹ . NE, using k, ε, reaction time (t seconds) and absorbance change (ΔA) up to the half-life of NEPO methyl ester (0.693 / k).
The initial concentration of PO methyl ester and the singlet oxygen concentration assuming that the formed singlet oxygen accumulates without deactivation can be calculated as follows. [NEPO methyl ester] ₀ = 2 ΔA / ε M (1) [ ¹ O ₂ ] = (1-exp- ^kt ) [NEPO methyl ester] ₀ M (2) Also contains 1% ethanol at 25 ° C. K in PBS was (8,34 ± 0.36) × 10 ⁻⁵ s ⁻¹ .

C. Demonstration that NEPO methyl ester is taken into C-26 cells (colorectal cancer cultured cells) and changes to NEPO NEPO methyl ester is very slowly decomposed at 0-4 ° C. The introduction was performed on ice. 5 × 10 ⁴ C-26 cells were cultured in 500 μl of culture medium under an oxygen atmosphere containing 5% CO _{2 for} 24 hours.
Incubate at 37 ° C. overnight in a well culture plate. Cultures were L-glutamine, 1% antibiotic-antifungal, and 10% fetal calf serum (Cancer International).
al Inc. Tokyo) (PRMC-1640 culture medium (GIBCOBRL, Tokyo)). Thereafter, the culture solution was removed with an aspirator, and the cells in the wells were washed with PBS, and then 237.5 μL of PBS and 12.5 μL of 20 m
After adding M NEP0 methyl ester, NEPO
Incubation was performed on ice so that P0 methyl ester was taken up into cells without thermal decomposition. The final concentration of NEP0 methyl ester in the well in this operation was 1 m
M, final ethanol concentration was less than 5%. On ice,
After incubation for 2.5, 5, 10, 20, 30, 45 or 60 minutes, respectively, the supernatant of each well was removed and washed with 500 μL of PBS. Add 0 .0 to each well.
50 μL of 25% EDTA-trypsin was added and the cells were pipetted into microtubes. Further, each well was washed with 200 μL of methanol, and the washing solution was kept at 37 ° C. for 24 hours according to the cells in the microtube using each micropipette. Each micropipette was shaken vigorously for 1 minute and then centrifuged at 12000 rpm for 3 minutes. 10 μL corresponding to 2000 cell extracts
Was injected into a high performance liquid chromatograph (HPLC) equipped with a fluorescence detector (manufactured by Hitachi: F1100), and NPA and NPA methyl ester were analyzed. Column is CA
PCELPAK C18 (4.6 × 250 nm, 5 μm, manufactured by Shiseido), and eluent was acetonitrile / methanol / water containing 1% acetic acid (= 20/19/1, volume ratio). The excitation and emission wavelengths of the fluorescence detector are 292 nm and 340 nm, respectively.
Met. The result of FIG. 3 was obtained from the above experiment. That is, in 2.5 minutes, 0.08 fmol of NEPO methyl ester was incorporated into one C26-cell. C-26
Since the amount of NEPO methyl ester uptake in the cells increased up to 45 minutes, the NEPO methyl ester uptake time in C-26 cells in the following experiments was set to 60 minutes. The maximum uptake of NEPO methyl ester in one C26-cell was 0.18 fmol.

D. Determination of viability of cells exposed to NEPO methyl ester C-26 cells in 1 mL of RPMI-1640 medium containing 10% fetal porcine plasma (corresponding to 1 × 10 ⁵ cells)
At 37 ° C. on a 24-well culture plate with 5% C
Incubated for 24 hours in an oxygen atmosphere containing O ₂ .
Then, after carefully removing the culture solution using an aspirator, the remaining cells were washed twice with 250 μL of PBS. In each well, add 12.5% NEPO methyl ester (NPA methyl ester or NEPO) ethanol solution.
μL and 237.5 μL of PBS were added, and the mixture was incubated at 0 ° C. for 60 minutes to incorporate NEPO methyl ester into the cells. Under these conditions, some of the NEPO methyl ester and NPA methyl ester are taken up into the cell and remain in the cell in the form of endoperoxide without releasing singlet oxygen at the low temperature.
After carefully removing the solution using an aspirator, the cells were washed with 250 μL of PBS. Then, 250 μL of PBS was added to the wells, and 15, 30, 45, 37 ° C. to expose the inside of the cells to singlet oxygen.
And incubated for 60 minutes. At the end of the incubation, cells were stained with trypan blue. For each sample, count 500 cells,
The ratio of viable cells (unstained cells) to total cells was expressed as a percentage of viability.

F. Measurement of viability of cells exposed to NEPO methyl ester by another method (using formazan formation from tetrazoline in cells exposed to NEPO methyl ester as an index) From the experiment evaluated by trypan blue staining described above, NEPO Methyl ester was found to be highly toxic to C-26 cells, further indicating that live cells form and formazane from tetrazoline, whereas dead cells do not. The following was confirmed using an assay method using A suspension in which 1 × 10 ⁵ C-26 cells were dispersed in 1 mL of an RPMI-1640 culture medium containing 10% fetal bovine plasma was prepared, and 100 μL of the suspension was dispensed into each well of a 96-well microtiter plate. 5% CO at 37 ° C
After incubation for 24 hours in an atmosphere of oxygen containing ₂ , the supernatant was carefully removed by aspirator. The cells remaining in each well were washed twice with 100 μL of PBS.
The final concentration of NEPO methyl ester in the well is 0.1-
To each well, 95 μL of PBS and 5 μL of a solution of NEPO methyl ester in ethanol were added to 1.0 mM. After incubating at 0 ° C. for 60 minutes, the supernatant was removed with an aspirator, and the remaining cells were washed with 100 μL of PBS.
Washed twice with. 100 μL of PBS in each well
And incubated at 37 ° C. for 1, 2, 3, 4, and 5 hours. At the end of the incubation, 10 μL of XTT reagent (Wako) was added to each well.

The XTT reagent is a novel tetrazolium, 2,
3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5-[(phenylamino) carbonyl] -2
It is an evaluation kit (cell count kit) manufactured by Wako that contains H-tetrazonium hydroxide).

After incubating the sample containing the above reagent at 37 ° C. for 3 hours, a microplate reader (microp
450nm derived from tetrazolium using a late reader)
Was measured for absorbance. 100% absorbance of control
Survival was calculated as the survival rate due to mitochondrial function as follows. Viability (%) = 100 (TB) / (UB) where T is tetrazolium when C-26 cells are exposed to NEPO methyl ester, U is control (untreated cells), B is the absorbance when neither NEPO methyl ester nor XTT is added.

E. C-26 cells to NEPO methyl ester
Exposure changes adenosine triphosphate (ATP) content in cells
Proof of what you do. R containing 10% fetal porcine plasma
1 x 10 in 1 mL of PMI-1640 culture ^FiveC-
Prepare a suspension containing 26 cells and apply it to 95 μL
Transfer to each well of the well plate and add 5% CO at 37 ° C._TwoIncluding
Incubated for 24 hours in an aerobic atmosphere. NEP
O-Methylester ethanol solution containing C-26 cells
In addition to each well of a 96-well plate, NEPO Methyl S
The ethanol solution of Ter was added to a final concentration of 0.1 mM, 0.2 mM.
mM, 0.5 mM and 1 mM.
At room temperature for 60 minutes and NEPO in the cells.
After incorporation of the methyl ester, the cell suspension was
Centrifuged at 50 g. Discard the supernatant and remove the precipitated cells
Re-disperse in 100 μL of PMI-1640 culture
Incubate at 7 ° C for 15, 30, 45 and 60 minutes
I was vate. Incubated for different times
Thereafter, the ATP concentration in the cells was measured by a luminescence method.
Was. Of C-26 cells exposed to NEPO methyl ester
ATP production is significantly reduced compared to control experiments.
I was Based on this fact, fineness by NEPO methyl ester
Vesicle damage is caused by singlet oxygen from NEPO of Escherichia coli
It turned out to be the same mechanism as disability.

G. NEP by leakage of butyrate dehydrogenase (LDH) from cells exposed to NEPO methyl ester
Evaluation of toxicity of O-methyl ester on cell membrane NEPO methyl ester attacked cell membrane.
Assuming that the cell cell membrane is destroyed and dies, butyrate dehydrogenase (LDH) dissolved in the cytosol flows into the extracellular fluid. Therefore, it was examined by the following experiment whether LDH flowed out of the cells exposed to NEPO methyl ester into the extracellular fluid. 1 × 10 ⁵ C-26 cells contained 10% fetal bovine plasma in RPMI-1640 culture 1
A 50 μL suspension was prepared in 96 mL.
Aliquots were added to each well of a well microtiter plate (Costar) and incubated at 37 ° C. under an oxygen atmosphere containing 5% CO ₂ for 24 hours, and then the supernatant was carefully removed by aspirator. Cells remaining in each well are removed using PBS.
After washing twice with 100 μL and cooling to 0 ° C., PBS 9
5 μL of NEPO methyl ester in ethanol 5
μL was added to each well. The final NEPO methyl ester concentration in the wells was between 0.1 mM and 1 mM. After incubating the cells at 0 ° C. for 60 minutes, the supernatant was removed with an aspirator and washed twice with 100 μL of PBS. Then, 100 μL of PBS was added to each well,
Incubated at 37 ° C. for 15, 30, 45 and 60 minutes, respectively. After incubation is completed,
Was centrifuged at 1,000 times / minute for 3 minutes. An LDH-cytotoxicity test WAKO (Wako Pure Chemical Industries, Osaka) was added to the supernatant, and the amount of LDH eluted outside the cells was quantified.

H. Singlet oxygen production and cell death in cells. (A) High concentration in cytoplasm exposed to NEPO methyl ester
Of NPA was detected and (b) exposed to NEPO methyl ester.
Exposure of dead cells to (c) mitochondria
ATP production was inhibited, and (d) cell plasma membrane
Is almost injured for three things:
Suggests. (1) NEPO methyl ester is intracellular
NEPO by the action of esterase in the cytoplasm
Is hydrolyzed to (2) NEPO generated is cytosol
And NPA and singlet acid thermally in a cytosol
Decomposed to elemental and inhibits (3) mitochondrial respiratory enzyme
And cause the cells to die. On the other hand, NEPO methyl S
Ter, in extracellular fluid, also contains NPA methyl ester and
It has been proven to be decomposed into doublet oxygen. At 0 ° C,
The final concentration of NEPO methyl ester should be 1 mM
2.1 × 10 ^Three237.7 μL of PBS containing cells
125 μL of NEPO methyl ester in ethanol
In addition, incubation was performed for a predetermined time. Wash cells, E
Treated with DTA-trypsin, then extracted with methanol
Was. The methanol extract was left overnight at 37 ° C.
C (High Performance Liquid Chromatograph) sample. FIG.
As shown in the figure, the HPLC chart shows three fluorescent peaks.
, Ie, unknown compounds, NPA and NPA methyl
A luster peak appeared. Using the HPLC method, 0
Exposure to NEPO methyl ester described in FIG.
Occurred in cell cytosol at time¹O_TwoThe amount of NPA
Can be estimated by measuring the amount of

[0021]

EXAMPLES Example 1 (a) Killing effect of NEPO methyl ester on C-26 cells evaluated by trypan blue staining Dead cells are stained with trypan blue but normal cells are not stained using the fact that they are stained. NE for 26 cells
The PO methyl ester toxicity test was performed as follows. At 0 ° C., 12.5 μL of a solution of NEPO methyl ester in ethanol was added to a sample containing 2.1 × 10 ³ cells in 237.5 μL of PBS to a predetermined final concentration of NEPO methyl ester, and the mixture was incubated for 60 minutes. PB
After washing with S, the temperature was maintained at 37 ° C. After a predetermined incubation time, the viability of the cells was evaluated by a trypan blue staining test. Cells (5 × 10 ⁵ /
(500 μL) was incubated at 0 ° C. for 60 minutes with PBS containing 1 mM NEPO methyl ester, and the cells washed with PBS were kept at 37 ° C. for a predetermined time. Immediately, cells were stained with 0.4% trypan blue and counted. For each sample, 500 cells were taken and live cells were expressed as viability (%). As is clear from the results summarized in FIG. 4, all the C-26 cells exposed to NEPO methyl ester at a concentration of 100 μM or more died within 1 hour. On the other hand, at a NEPO methyl ester concentration of 5 μM or less, cells have little effect within 5 hours. C-26 cell viability after 5 hours is 90 μm per hour at 10 μM.
%, 5% at 65%, and 50% at 50 μM.
Thus, it was revealed that NEPO methyl ester is strongly toxic to C-26 cells. The prolonged cytotoxicity due to low concentrations of NEPO methyl ester is presumed to be due to apoptosis.

(B) Evaluation of cell killing effect by measuring the ability to form formazan from tetrazoline in cells exposed to NEPO methyl ester XTT (tetrazolium reagent) is nontoxic and soluble by metabolic reduction by living cells. Designed to produce colored formazan. The tetrazolium ring is opened by dehydrogenation in particularly active mitochondria, so the reaction only takes place in active mitochondria and in living cells. Thus, the method can assess cytotoxicity associated with impaired mitochondrial function.

1 × 10 ⁴ cells in 95 μL of PBS were exposed to NEPO methyl ester at 0 ° C. for 60 minutes, and the cells were washed with PBS and incubated at 37 ° C. for the times indicated. At the end of the incubation, the tetrazolium reagent is added,
Incubation was further performed at 37 ° C. for 3 hours. The formation of formazan was determined from the absorbance. The results obtained are shown in FIG. In this experimental method, NEPO with a concentration of 100 μM or more was used.
C-26 cells exposed to methyl ester confirmed that all died within one hour. These results support that the cytotoxicity of NEPO methyl ester is due to mitochondrial damage. In this method, only 10μM NEPO
Over 9% of C-26 cells exposed to methyl ester were found to be injured within 5 hours.

(C) Evaluation of NEPO methyl ester toxicity on cell membranes It was confirmed by the following experiment whether or not NEPO methyl ester exerts toxicity on cell membranes during its uptake into cells. In other words, it was proved that the killing of cells by NEPO methyl ester resulted from intracellular organ damage due to generation of singlet oxygen in the cells. In this experiment, PB
5 × 10 ³ cells in 95 μL of S were exposed to 5 μL of NEPO methyl ester at 0 ° C. for 1 hour. After washing with PBS, the cells were incubated at 37 ° C. for a predetermined time. At the end of the incubation, LDH activity was measured. FIG. 6 shows that LDH release from cells exposed to 100-1 μM NEPO methyl ester was 0.2%
Treatment with Tween 20, which has a cytoplasmic membrane disrupting action at a high concentration, causes LD when all cell membranes are destroyed.
This indicates that the amount is very small as compared with the amount of released H (100%). As is clear from the results shown in FIG.
No LDH was detected in the cell solution of the cells exposed to 0 μM NEPO methyl ester cultured at 37 ° C. for 1 hour.
Since most of the C-26 cells (colorectal cancer cultured cells) have been killed under these conditions, it is clear that the cell death due to NEPO methyl ester is not due to the disruption of the plasma membrane. These observations, ¹ O ₂ generated in the cells generated in the extracellular ¹
As with O ₂ , it shows almost no damage to cell membranes. That is, this indicates that the compound of the present invention is effectively taken into cells and functions effectively in killing cells in cells.

Example 2 Killing Experiment of Rat Thoracic Aortic Smooth Muscle Cells with NEPO Methyl Ester A thoracic aorta was quickly taken out from an ether-anaesthetized male Wistar rat, and blood cells were washed out in Krebs-Ringer solution, and then divided into 8 equal parts (about 5 parts). mm), and endothelial cells were gently rubbed with soft paper in a Krebs-Ringer solution under a stereoscopic microscope. Each specimen was cut into a spiral shape and stored in an ice bath in the Krebs-Ringer solution. Immediately at 37 ° C, bubble 20% oxygen gas containing 5% carbon dioxide.
One end of the specimen was tied with a silk thread in a container filled with Krebs-Ringer solution, and one end of the thread was fixed on an acrylic rod. Similarly, the other end was fixed to the transducer with a silk thread. The amplifier was adjusted so that 1 g was 2.5 cm on the recording paper. A 0.4 g tension was applied to the sample, and when the tension became stable, the Krebs-Ringer solution was replaced with a new one.After the sample was contracted by the Krebs-Ringer solution with a high potassium ion concentration and the tension was stabilized, the solution was replaced with the Krebs-Ringer solution. Relaxed. After repeating potassium ion-dependent muscle contraction-relaxation by Krebs-Ringer washing, the compound was added so that the final NEPO methyl ester concentration in the bath was 100 mM. After washing twice with the Krebs-Ringer solution, 13 minutes later, methoxamine, a smooth muscle contraction agent, was added to a concentration of 50 mM to shrink the sample. Nearly normal tension was obtained, indicating at this point that the specimen received minimal damage. When the tension was stabilized after 8 minutes, when the compound was added so that the final NEPO methyl ester concentration in the Krebs-Ringer solution in the bath became 770 mM, the tension gradually started to decrease, and after 40 minutes, 60% Down to. After washing twice with Krebs-Ringer solution, only a 10% recovery in tension was observed. When a similar experiment was performed using 2.3 mM NEPO ester, the muscle tension decreased sharply, and after 20 minutes, completely decreased to the tension before the administration of methoxamine.
This indicates that NEPO methyl ester seriously injured the rat thoracic aorta specimen and abolished its function.

Application Example From the above, ethanol or PBS containing 1% ethanol was used as a solvent, and 0.1 to 1 mM NEPO was added thereto.
It can be inferred that an injection solution containing a methyl ester is prepared, and the injection solution is injected into a milk duct of a breast cancer to kill breast cancer cells. Since the breast cancer is formed on the surface of the endothelium of the mammary gland, it can be easily predicted that the breast cancer cells can be killed with high efficiency by absorbing NEPO methyl ester by contact with the injection solution. In addition, it can be easily inferred from the results of the live cell killing experiment that the cancer cells could be killed even when the injection solution was injected into a liver cancer patient. The cell-invasive singlet oxygen generator may also work effectively for other cancers.
Although this injection solution cannot specifically act on cancer cells and kills normal cells, the half-life of NEPO methyl ester is as short as 30 minutes, which is a great advantage that does not affect the growth of normal cells after treatment. There is.

The injection solution is sustained-released by a carrier such as a porous capsule [Millipore chamber (diameter 10 mm, thickness 2 mm).
mm, filter pore size 0.22 μm] and apply to the affected area.

[0028]

As described above, the compounds of the present invention are:
It is efficiently taken up into eukaryotic cells, changes into a cytosolic soluble singlet oxygen generator in the cell, and has an excellent effect of effectively generating singlet oxygen by using body temperature as a trigger. As a result, mitochondria are damaged and eukaryotic cells die.

[Brief description of the drawings]

FIG. 1. 37% PBS in 1% ethanol.
Change with time in NEPO methyl ester decomposition at 80 ° C (NPA methyl ester formation process, absorption spectrum 28
(Track 8nm)

FIG. 2. C exposed to 1 mM NEPO methyl ester
-26 High performance liquid chromatogram of intracellular products, monitor by fluorescence.

FIG. 3. Time-dependent change in the amount of NEPO methyl ester introduced into C-26 cells

FIG. 4: Viability of cells exposed to NEPO methyl ester (evaluated by trypan blue staining)

FIG. 5: Correlation between NEPO methyl ester concentration and cell killing (evaluation with XTT reagent)

FIG. 6: Examination of the presence or absence of cell membrane damage due to NEPO methyl ester (evaluation based on the outflow of LDH outside the cell)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 35/00 C07D 493/18 C07D 493/18 // C01B 13/02 C01B 13/02 Z F-term (reference) 4C071 AA03 AA07 AA08 BB01 BB02 CC12 CC14 EE04 EE06 FF16 GG01 KK01 LL01 4C076 AA12 BB11 BB32 CC27 CC41 EE56M EE56N FF32 FF34 FF68 4C086 AA01 AA02 NA04 MA04 NA14

Claims (4)

[Claims] 1. A cell-penetrating excited singlet oxygen-generating compound represented by the general formulas A, B and C. Embedded image In the above general formulas A, B and C, m and n are 0,
1 R ₁ or 2 selected from the compounds of formula A,,
R ₂ , R ₅ and R ₆ are H, lower hydroxyalkylene group, lower alkoxy group, CN or COOR ₂₁ (provided that
R ₂₁ is selected from lower alkyl group), at least one is COOR _21, R ₉ in the general formula B, R _{1 0,} R
₁₃ and R ₁₄ are selected from H, lower hydroxyalkylene group, lower alkoxy group, CN or COOR ₂₁ (where R ₂₁ is a lower alkyl group), and at least one is COO
R ₂₁ and R ₁₅ to R ₁₈ in the general formula C are H, a lower hydroxyalkylene group, a lower alkoxy group,
CN or COOR ₂₁ (however, R ₂₁ is a lower alkyl group)
Wherein at least one is COOR ₂₁ and R
₃ , R ₄ , R ₇ , R ₈ , R ₁₁ , R ₁₂ , R ₁₉ and R ₂₀ are H,
It is a group selected from a lower alkyl group, a lower alkoxy group, a lower hydroxyalkylene group and CH ₂ COOR ₂₂ . 2. A cell-invasive drug which generates singlet oxygen at body temperature, comprising a compound represented by the general formulas A, B and C. 3. An injection comprising the compounds represented by the general formulas A, B and C. 4. A drug comprising a compound represented by the general formulas A, B and C, wherein the drug is held on a sustained-release carrier.